JP2009085813A - Immunochromatographic testpiece and measurement method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunochromatographic testpiece easily operable and capable of high-sensitivity immunochromatographic measurements, and to provide an immunochromatography measurement method using the same. <P>SOLUTION: An immunochromatography measurement system comprises a means for connecting the immunochromatographic testpiece 2, in such a way as to pass electricity; a means for detecting the passage of a mixed reagent of a first antibody, enzyme-carrying insoluble particles, and pigments through a second antibody immobilizing part of the immunochromatographic testpiece 2; a means for applying a voltage to electrodes of the immunochromatographic testpiece; and a means for measuring changes by immunoreaction at the second antibody immobilizing part of the immunochromatographic testpiece 2. After the passage of the mixed reagent of the first antibody, the enzyme-carrying insoluble particles, and the pigments through the second antibody immobilizing part is detected, and the discharge of a substrate solution from an electrically responsive gel is accelerated by applying a voltage to the electrodes of the immunochromatographic testpiece 2, and the operation of measuring changes by immunoreaction at the second antibody immobilizing part of the immunochromatographic testpiece 2 is performed. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an immunochromatographic test piece for measuring the amount of a specific substance contained in a collected body fluid and an immunochromatographic measurement method using the same.

  Conventional measuring instruments used in the clinical laboratory field include large automated instruments such as general-purpose biochemical analyzers capable of multi-item biochemical examinations, and relatively small instruments used for immediate examinations at medical examination sites. There is a POCT (Point Of Care Testing) inspection device.

  The above-mentioned large-scale automated equipment is installed in central clinical laboratory departments and clinical laboratory contract companies in hospitals where it is necessary to conduct multi-item examinations of a large number of patient specimens. Examples of testing equipment used in home medical care include a blood glucose sensor that detects an enzyme reaction, a pregnancy diagnostic agent that detects an immune reaction, and an ovulation test agent. Since the POCT test equipment is used for screening and monitoring of the medical condition at home and at home, rapid measurement is possible, and it is assumed that it is operated by a person who is not a test specialist, and in terms of operability, There is a need for simplicity that can be used by anyone without special expertise.

  A typical example of a widely used POCT testing device is a pregnancy diagnostic agent. This is based on the immunochromatography method.

  The typical structure of the equipment used for the immunochromatography method is as follows. First, a developing device using a nitrocellulose membrane or the like cut into an elongated rectangular shape is used as the sample liquid developing portion. A pad made of cellulose or the like as a material is disposed as an appropriate lower part of the sample solution at a position in contact with one end of the sample solution developing unit so that the sample solution flows along the long side of the sample solution developing unit. Downstream of the lower part of the sample droplet, a filter paper that has been impregnated with an antibody that specifically binds to an antigen in the sample liquid and labeled with particles such as gold colloid and then dried is used as a labeled reagent part. It arrange | positions so that a liquid expansion | deployment part may be contact | connected. An antibody that binds to an antigen is immobilized on the sample solution developing section downstream of the labeling reagent section as a detection section at a location different from the labeled antibody. A pad made of cellulose or the like as a material is disposed downstream of the detection unit as a sample solution absorption unit so as to contact the remaining end of the sample solution development unit.

  By dropping the sample liquid onto the lower part of the sample droplet of the equipment obtained in this way, the sample liquid moved by the capillary phenomenon in the sample liquid developing part is in the sample liquid labeled with particles such as gold colloid in the labeling reagent part. A reagent containing an antibody that specifically binds to an antigen is dissolved, and the antibody that specifically binds to the antigen in the sample solution labeled with particles such as the dissolved gold colloid reacts with the antigen in the sample solution, and the conjugate of the antibody and antigen becomes Arise. Due to the capillary action, the bound substance further moves downstream in the sample liquid development part, and the antigen that binds to the antigen in a different location from the labeled antibody immobilized on the bound part of the bound antigen and the detection part causes a binding reaction, In the detection unit, a sandwich-like conjugate is formed by the immobilized antibody, antigen, and labeled antibody, and when the antigen is present in the sample solution, a change depending on the label can be detected in the detection unit. For example, when the marker is colored, it can be grasped as a color change by visual recognition or a change in absorbance by a spectroscope. Further, the excess sample solution and reagent moved downstream from the detection unit are absorbed by the pad of the sample solution absorption unit.

In order to increase the sensitivity of such an immunochromatography method, an sensitization method using an enzyme is shown, and an enzyme is used in place of particles as a label for an antibody having the above-mentioned equipment structure, and a substrate for an enzyme reaction. Is known to be included in a developing solution, or a substrate is impregnated in a pad of a sample dropping part (see, for example, Patent Document 1). In addition, instead of the sample solution developing part having the above-described equipment configuration, a dropping pad for dropping the developing solution for the enzyme reaction is provided, the labeling reagent part is eliminated, and the sample solution and the labeled antibody are disposed downstream of the dropping pad. There is known one in which a dropping pad for dropping a liquid reagent having an enzyme immobilized thereon is further provided on the surface of the labeled particle (see, for example, Patent Document 2).
JP-A-11-153600 JP 2001-13144 A

  However, in the conventional highly sensitive immunochromatographic test piece shown in Patent Document 1, two steps of supplying a sample solution and supplying a developing solution for the enzyme reaction are performed by the user at the time of measurement. In addition, in Patent Document 2, a step of supplying a sample solution, a step of supplying a liquid reagent in which an enzyme is further immobilized on the surface of a labeled particle of a labeled antibody, and a step of supplying a developing solution for an enzyme reaction In order to ask the user for these three steps, there is a problem that the operation is complicated. Further, when the liquid supply process is wrong, for example, when the developing solution for the enzyme reaction is supplied first, there is a problem that the enzyme reaction finally occurring in the detection unit becomes insufficient.

  The conventional problems as described above are assumed to be operated by a person who is not an expert, and will be further improved in the future in POCT inspection equipment that is required to be usable by anyone without requiring special expertise in terms of operability. Considering the spread, it is expected to be resolved immediately.

  Therefore, the present invention has an object to provide an immunochromatographic test piece that can solve the conventional problems as described above and can perform an immunochromatographic measurement that is easy to operate and highly sensitive, and an immunochromatographic measuring method using the same. .

  In order to achieve the above object, an immunochromatographic test piece in the present invention is an immunochromatographic test piece for measuring an antigen in a sample solution, which is an insulating substrate, a pair of electrodes disposed on the substrate, An electrically responsive gel disposed on a pair of electrodes and holding a solution containing a substrate for an enzyme, a porous member disposed on the substrate and in a position in contact with the electrically responsive gel, a first for the antigen A reagent part arranged on the porous member so that the reagent containing the antibody and the enzyme-carrying particles and the dye move together with the sample solution, the antibody against the antigen, A second antibody capable of binding to a site different from the antibody is disposed at a position where the second antibody is immobilized on the porous member and a position in contact with the porous member, and can absorb the sample liquid The reagent part, the immobilization part and the water absorption part are provided with a water part, and the sample liquid supplied on the porous member flows in the order of the reagent part, the immobilization part and the water absorption part. Has been placed.

Moreover, the immunochromatographic measurement method in the present invention uses the immunochromatographic test piece,
(A) supplying the sample solution onto the porous member;
(B) a step of detecting that the sample solution containing the reagent has passed through the immobilization unit;
(C) a step of automatically applying a voltage between the pair of electrodes based on the detection in the step B;
(D) detecting the amount of the reaction by the enzyme in the immobilization section; and (E) determining the concentration of the antigen contained in the sample solution based on the detection amount in the step D.

  According to the immunochromatographic test piece of the present invention and the immunochromatographic measurement method using the same, the immunochromatographic measurement sensitized by the enzyme can be performed only by the step of dropping the sample liquid onto the immunochromatographic test piece, without depending on the skill of the user. In addition, correct measurement can be performed.

  Embodiments of the present invention will be described below with reference to the drawings.

(Embodiment)
1 to 8 are diagrams for explaining an immunochromatography measurement system according to an embodiment of the present invention. FIG. 1 is a perspective view showing an appearance of the immunochromatography measurement system. FIG. 2 is a perspective view showing a test piece. 3 (a) is a side view showing the test piece, FIG. 3 (b) is a plan view showing the test piece, FIGS. 4 and 5 are process diagrams for each production process for explaining the configuration of the test piece, and FIG. FIG. 7 is a front view showing the structure of the measuring device seen partially broken, FIG. 7 is a cross-sectional view showing the structure of the measuring device taken along the line AA in FIG. 6, and FIG. FIG. 9 is a sectional view of the immunochromatographic measurement system when the test piece is at the measurement position, FIG. 10 is an operation timing explanatory diagram of the immunochromatographic measurement system, and FIG. 11 is a measurement. Block of processing control unit in apparatus It is shown.

  In FIG. 1, an immunochromatographic measurement system 1 includes a test piece 2 and a measuring device 3. A main power source 4, a display unit 5, a measurement start button 6, and a test piece insertion port 7 are disposed on the front surface of the measuring device 3. The measurement can be started by turning on the main power supply 4 and inserting the test piece 2 into the test piece insertion port 7 in accordance with the instruction of the display unit 5.

  First, the configuration of the test piece 2 will be described with reference to the drawings.

  As shown in FIGS. 2, 3 (a), and 3 (b), the test piece 2 includes an upper cover 21, a lower cover 22, and an inspection base 23. The upper cover 21 is provided with a sample liquid inlet 21a, a measurement window 21b for optical measurement, and an air hole 21c that is an air outlet considering expansion of the water absorption pad. By attaching the inspection base 23 between the upper cover 21 and the lower cover 22 and overlapping the upper cover 21 and the lower cover 22, the three side surfaces are closed, but the inspection is performed from the side surface at the other end. A part of the base 23 protrudes.

  Next, the configuration and manufacturing process of the inspection substrate 23 will be described.

  In the first step, as shown in FIG. 4A, a wiring part 42 made of copper foil is produced on a PTFE substrate 41 (for example, 1 cm wide × 10 cm long, 3 mm thick) by a known technique such as etching. To do.

  The wiring part 42 corresponds to the pair of connection parts 43a and 44a formed at one end part in the length direction, the electrode parts 43b and 43c corresponding to the connection part 43a in the vicinity of the other end part, and the connection part 44a. It consists of electrode portions 44b and 44c, a lead portion 43d that connects the connecting portion 43a and the electrode portions 43b and 43c, and a lead portion 44d that connects the connecting portion 44a and the electrode portions 44b and 44c.

  Then, the electrode part 43b and the electrode part 44b, and the electrode part 43c and the electrode part 44c are arranged so as to face each other. Copper, gold, platinum, or the like can be used as the material used as the wiring portion 42, but copper is preferable because it is relatively inexpensive.

  As the second step, as shown in FIG. 4 (b), a carbon film is formed on each of the electrode parts 43b, 43c, 44b and 44c and coated to form the carbon electrode parts 45b, 45c, 46b and 46c, respectively. To do. In addition to carbon, copper, gold, platinum, palladium, or the like can be used as a material used as the carbon electrode portions 45b, 45c, 46b, and 46c.

  As the third step, as shown in FIG. 4 (c), the carbon electrode portions 45b, 45c, 46b and 46c, the connection portions 43a and 44a, and a part of the lead portions 43d and 44d connected thereto are left. The surface of the other part is coated with a polyvinyl resin to form an insulating film 47.

As a fourth step, as shown in FIG. 4D, 3,3′-diaminobenzidine-containing polyvinyl alcohol is formed on a pair of carbon electrode portions 45b and 46b formed on the PTFE substrate 41 so as to cover them. The first electro-responsive gel 48 made of polyacrylic acid gel is adhered with spray glue; Further, from the polyvinyl alcohol-polyacrylic acid gel containing H ₂ O ₂ so as to cover the other pair of carbon electrode portions 45c and 46c so as to be adjacent to the first electroresponsive gel 48. The second electrically responsive gel 49 is adhered with spray glue.

The first electroresponsive gel 48 is prepared by adding a TBS buffer solution (0.05 M Tris-HCl, 0.15 M NaCl, pH 7.5) containing 0.5 mg / ml 3,3′-diaminobenzidine. A 3,3′-diaminobenzidine-containing polyvinyl alcohol-polyacrylic acid gel can be prepared and obtained by a sol-gel method using polyvinyl alcohol-polyacrylic acid. The second electrically responsive gel 49 in a solution by adding TBS buffer containing _0.15% H 2 _{O 2,} polyvinyl alcohol - sol with polyacrylic acid - gel _{method, H} 2 O ₂ containing polyvinyl alcohol-polyacrylic acid gel.

  As a fifth step, as shown in FIG. 5A, a nitrocellulose membrane 51 (for example, a thickness of 200 μm) is pasted on the insulating film 47 formed on the PTFE substrate 41 with spray glue. At this time, one end of the nitrocellulose membrane 51 is in contact with the end of the first electro-responsive gel 48 and the second electro-responsive gel 49 on the side of the connecting portions 43a and 44a, and the other end is insulated. The film 47 is formed so as to be in the vicinity of the end portions on the connection portions 43a and 44a side. Here, the nitrocellulose membrane 51 corresponds to the porous member in the present invention.

  The nitrocellulose membrane 51 used for the test substrate 23 of the test piece 2 is preferably subjected to a blocking treatment with a substance that does not participate in the antigen-antibody reaction after the second antibody described later is immobilized. In this way, it is possible to prevent nonspecific adsorption of the reagent containing the reagent on the nitrocellulose membrane 51 and the antigen in the sample solution to the nitrocellulose membrane 51, and to keep the background low. At the same time, it is possible to ensure the mobility of the mixed reagent of the first antibody and enzyme-supporting insoluble particles and dye described later supported on the nitrocellulose membrane 51.

For example, in this embodiment, in order to prevent nonspecific protein adsorption to the nitrocellulose membrane 51, a PBS buffer solution (8 g / l NaCl, 0.2 g / l KCl containing 1% by weight bovine serum albumin) is used. 1.15 g / l Na ₂ HPO ₄ · 12H ₂ O, 0.2 g / l KH ₂ PO ₄ , pH 7.4) for about 15 minutes and dried at 40 ° C. for about 2 hours to block the surface. Nitrocellulose membrane 51 is used.

  As a sixth step, as shown in FIG. 5 (b), the nitrocellulose in which the first reagent-supporting portion 52 composed of the first antibody, the enzyme-supporting insoluble particles and the phenanthroline iron complex as the dye is blocked is used. Formed on the membrane 51. The first antibody at a predetermined position of the nitrocellulose membrane 51 (for example, a position of about 1.5 cm from the end of the nitrocellulose membrane 51 on the side of the first electroresponsive gel 48 and the second electroresponsive gel 49). The first antibody, the enzyme-carrying insoluble particles and the phenance are prepared by applying a quantity of several μl of the suspension containing the enzyme-carrying insoluble particles and the dye phenanthrolin iron complex in a line by a dispenser and freeze-drying. The lorin iron complex can be fixed to the nitrocellulose membrane 51, and the first reagent carrying part 52 can be formed. Here, the first reagent carrying part 52 corresponds to the reagent part in the present invention.

  The first antibody, enzyme-supporting insoluble particles, and phenanthroline iron complex can be prepared, but commercially available ones can also be used. A construction method using commercially available polystyrene latex particles having a particle size of 0.2 μm will be described. 0.5 ml of 2.5% by weight polystyrene latex particles suspended in distilled water is placed in a microtube, and 0.02M boric acid, pH 8.2 buffer containing 1 mg / ml of the first antibody is added to the microtube. Add 5 ml and stir for 2 hours at room temperature.

  After stirring, the mixture was centrifuged at 12,000 rpm for 30 minutes to precipitate insoluble magnetic particles, and then the supernatant was removed. Subsequently, 0.01 M boric acid containing 0.5 mg / ml alkaline phosphatase, pH 8.2 buffer was added. 1 ml of the liquid is added to suspend the polystyrene latex particles, and the mixture is shaken and stirred at room temperature for 2 hours. After stirring, the mixture was centrifuged at 12,000 rpm for 30 minutes to precipitate insoluble magnetic particles, and then the supernatant was removed. Polystyrene latex with 0.01 M boric acid, 0.1 mM phenanthroline iron complex, pH 8.2 buffer solution By suspending and diluting so that the particle concentration is 2.5% by weight, the first antibody, the enzyme-supported polystyrene latex particles, and the phenanthroline iron complex can be obtained.

  As a seventh step, as shown in FIG. 5 (c), a second reagent carrying portion 53 constituted by the second antibody is formed on the nitrocellulose membrane 51. An aqueous solution in which 1 mg / ml to several mg / ml of the second antibody is dissolved at a predetermined position on the nitrocellulose membrane 51 (for example, a position of about 3 cm from the end of the connecting portion 43a and 44a in the nitrocellulose membrane 51). Is applied in a line of several μl with a dispenser and air-dried, whereby the second antibody can be fixed to the nitrocellulose membrane 51 and the second reagent-carrying part 53 can be formed. The second antibody is an antibody against an antigen and can bind to a site different from the first antibody. Here, the second reagent carrying part 53 corresponds to the immobilization part in the present invention.

  As an eighth step, the glass fiber absorbent pad 54 is placed on a predetermined position on the nitrocellulose membrane 51 (for example, a position 1.5 cm away from the second reagent carrying portion 53 toward the connecting portions 43a and 44a). Apply with spray glue. Further, the sample liquid filter paper 55 is disposed on the nitrocellulose membrane 51 with a spray paste between the first reagent carrier 52 and the first and second electroresponsive gels 48 and 49. Thus, the inspection substrate 23 is completed. Here, the water absorbing pad 54 corresponds to the water absorbing portion in the present invention.

  The test piece 2 can be manufactured by mounting the upper cover 21 and the lower cover 22 on the test base 23 manufactured through the above steps. When the upper cover 21 is attached to the inspection base 23, the air holes 21 c provided in the upper cover 21 are disposed at positions corresponding to the water absorption pads 54 of the inspection base 23, and the sample liquid filter paper 55 of the inspection base 23 is provided. A sample solution inlet 21a provided in the upper cover 21 is disposed at a position corresponding to the above.

  As described in the present embodiment, it is desirable to place the test piece 2 in a case made up of the upper cover 21 and the lower cover 22. In that case, although not described or illustrated in the present embodiment, it is preferable to provide a confirmation window so that the second reagent carrying part can be confirmed in order to maintain the visibility of the reaction. This confirmation window is substantially flat and is preferably sealed with an optically substantially transparent material such as polystyrene or quartz glass.

  Next, the configuration of the measuring device 3 will be described with reference to the drawings. As shown in FIG. 1, the measuring device 3 has a main power supply 4, a display unit 5, a measurement start button 6, and a test piece insertion port 7 in its front part. In addition, the measuring device 3 includes a processing control unit 60 having a calculation unit, a time measuring unit, a memory, a ROM, and a pulse counter in order to perform measurement operation of the test piece 2 and analysis of the results.

  The measuring device 3 has an optical measurement chamber for detecting and measuring a reaction generated in the test piece 2 inside.

  6 and 7, the optical measurement chamber includes a measurement chamber casing 62 attached to an apparatus casing 61 that constitutes the measuring device 3, a casing 63 that constitutes the measurement chamber casing 62, A table portion 64 that linearly moves in a space formed in the housing 63 and a drive mechanism portion 65 for linearly moving the table portion 64 are provided.

  The measurement chamber casing 62 includes a casing 63 and an optical measurement unit 66. The housing 63 includes a housing upper portion 63a, side surface portions 63b formed on both sides of the housing upper portion 63a, and a reinforcing portion 63c that reinforces the side surface portion 63b by connecting the lower portions of the side surface portions 63b.

  The housing upper part 63a is provided with an optical measurement attachment part 63d having two inclined surfaces and attaching the optical measurement part 66, and the cross-sectional shape of each side face part 63b is opposite to the opposite side surface 63b. A letter-shaped groove 63 e is arranged in parallel to the longitudinal direction of the housing 63.

  The optical measurement unit 66 includes a blue light source 67, an optical slit 68, and a detector 69. The blue light source 67 and the optical slit 68 are attached to one inclined surface of the optical measurement attachment portion 63d of the housing upper part 63a.

  The detector 69 is attached to the other inclined surface of the optical measurement attachment portion 63d. The light emitted from the blue light source 67 is transmitted through the optical slit 68 so that it is perpendicular to the length direction of the test piece 2 (not shown) attached at a predetermined position for inspection measurement. adjust.

  The blue light source 67, the optical slit 68, and the detector 69 are irradiated with light emitted from the blue light source 67 through the optical slit 68 into the measurement window 21 b of the test piece 2, and the irradiated light is reflected by the test piece 2. The reflected light is arranged so that it can be captured by the detector 69.

  The table portion 64 includes a table body 71, a connector portion 72, and a driven screw portion 73. The table main body 71 has a holding surface 71a on which the test piece 2 is placed, a guide surface 71b that is perpendicular to the holding surface 71a and that faces both sides in order to guide the test piece 2 to a predetermined position, and a holding surface 71a. On the opposite side of the guide surface 71b, there are two side surfaces 71c perpendicular to the holding surface 71a, and the outer surface of each side surface 71c is substantially U-shaped parallel to the longitudinal direction of the holding surface 71a. A protrusion 71d is provided.

  The connector 72, which is formed by molding the contact piece 72a corresponding to the connection portions 43a and 44a in the above-described test piece 2 with the housing 72b, is fixed to a predetermined position of the table body 71. Further, in the recess 71e surrounded by the holding surface 71a of the table main body 71 and the two side surface portions 71c provided with the projections 71d, the driven screw portion 73 in which the female screw nut portion 75 is fixed to the bearing housing 74 is the table main body 71. It is fixed to.

  The drive mechanism 65 includes a stepping motor 65a attached directly or indirectly to the apparatus housing 61 and a male screw portion 65b fixed to the rotation shaft of the stepping motor 65a.

  The male screw portion 65b is screwed with the female screw nut portion 75 of the driven screw portion 73 in the table portion 64, and the female screw nut portion 75 is linearly moved by the rotation of the male screw portion 65b accompanying the rotation of the stepping motor 65a. Therefore, since the projection 71d of the table main body 71 connected to the female screw nut portion 75 is slidably fitted in the groove 63e provided in the housing 63 of the measurement chamber housing portion 62, the table main body 71 is It can move linearly along the groove 63e.

  The operation of the immunochromatographic measurement system including the test piece 2 and the measuring device 3 will be described with reference to FIGS.

  The main power supply 4 of the measuring apparatus 3 shown in FIG. 11 is turned on. By turning on the main power supply 4, a signal is transmitted to the calculation unit 101 of the processing control unit 60 of the measuring device 3, and the blue light source 67 is turned on by a signal from the calculation unit 101 corresponding thereto. Further, a signal for displaying a message for requesting insertion of the test piece 2 into the test piece insertion port 7 is sent from the calculation unit 101 to the display mechanism unit 102 including the display unit 5, a display IC, and a driver circuit. A display for instructing insertion of the test piece 2 is displayed on the display unit 5.

  As shown in FIG. 8, the test piece 2 is inserted from the test piece insertion port 7 along the holding surface 71 a and the guide surface 71 b of the table main body 71 according to the instruction of the display unit 5. Then, the connection portions 43 a and 44 a of the PTFE substrate 41 in the inspection base 23 of the inspection piece 2 are inserted so as to be held by the contact piece 72 a of the connector portion 72 and the housing 72 b of the measuring device 3. At this time, the test piece 2 is set to the initial position, and the sample liquid injection port 21 a of the test piece 2 is at least outside the device housing 61 of the measuring device 3. Further, the pulling force for pulling out the PTFE substrate 41 from the connector portion 72, that is, the pulling force may be set from several hundred grams to several kilograms.

  After inserting the test piece 2 into the measuring device 3, the measurement start button 6 is turned on.

  Then, the calculation unit 101 shown in FIG. 11 receives the measurement start signal from the measurement start button 6, and the calculation unit 101 instructs the stepping motor 65 a of the drive mechanism unit 65 to move the table unit 64 to a predetermined measurement position. To do. At this time, as shown in FIG. 9, the light emitted from the blue light source 67 and irradiating the test piece 2 through the optical slit 68 passes through the measurement window 21 b provided in the upper cover 21 of the test piece 2. The test piece 2 is held at a measurement position where the irradiated and reflected light reaches the detector 69 through the measurement window 21b.

  After the table unit 64 has moved to a predetermined measurement position, the calculation unit 101 irradiates the light emitted from the blue light source 67 through the measurement window 21b of the test piece 2 to the vicinity of the second reagent carrying unit 53. Then, a signal from the detector 69 that detects the reflected light is detected.

  The detection signal from the detector 69 is AD converted by the calculation unit 101, timed by the timing unit 103 provided in the processing control unit 60, and the detection signal AD-converted at 0.5s intervals is calculated by the calculation unit. Starting to be imported to 101, the acquired numerical value is stored in the memory 104 of the processing control unit 60.

  Further, the captured numerical value is compared with a threshold value I stored in advance in the ROM 105 of the processing control unit 60 by the calculation unit 101 as shown in FIG. When the captured numerical value exceeds the threshold value I, the arithmetic unit 101 instructs the stepping motor 65a of the drive mechanism unit 65 to return the test piece 2 placed on the table unit 64 to the initial position.

  In FIG. 8, after returning the test piece 2 to the initial position, a signal for displaying a message instructing to inject the sample liquid from the sample liquid injection port 21 a is sent from the calculation unit 101 to the display mechanism unit 102. A display instructing the sample solution injection is displayed on the display unit 5.

  In accordance with the instruction of the display unit 5, the sample liquid collected in a receptacle such as a cup is sucked into the dispenser, and a predetermined amount of the sample liquid sucked into the dispenser is injected from the sample liquid injection port 21a of the test piece 2.

  In the immunochromatography measurement system of the present invention, examples of the sample liquid include urine, blood, plasma, serum, saliva, cell interstitial fluid, sweat, tears, and the like. Examples of the antigen in the sample solution include haptens, proteins, DNA, bacteria, viruses and the like.

  Then, the calculation unit 101 instructs the stepping motor 65a of the drive mechanism unit 65 to return the test piece 2 placed on the table unit 64 to the measurement position again.

  In FIG. 9, the sample liquid injected into the test piece 2 passes through the sample liquid filter paper 55 of the test piece 2 inside the sample liquid injection port 21a, moves the nitrocellulose membrane 51 by capillary action, and the first reagent carrying part. 52 is reached. The sample solution that has reached the first reagent-carrying part 52 dissolves the first antibody, the enzyme-carrying polystyrene latex particles, and the phenanthroline iron complex that constitute the first reagent-carrying part 52.

  When the dissolved first antibody and the enzyme-supported polystyrene latex particles have an antigen in the sample solution, the antigen and the first antibody on the polystyrene latex particles cause a binding reaction. The combined substance of the antigen, the first antibody and the enzyme-supported polystyrene latex particle and the phenanthroline iron complex further move through the nitrocellulose membrane 51 by capillary action and reach the second reagent-supporting portion 53. When the antigen is present in the sample solution, the first antibody and the enzyme-supported polystyrene latex particles are captured by the binding between the second antibody and the antigen.

  The first antibody and the enzyme-carrying polystyrene latex particles that are not captured by the second reagent-carrying part 53 further move through the nitrocellulose membrane 51 and are absorbed by the water-absorbing pad 54 by capillary action.

  As described above, the sample solution that has passed through the first reagent-carrying part 52 contains the first antibody, which is a reagent, the enzyme-carrying polystyrene latex particles, and the phenanthroline iron complex dissolved therein. When the sample solution containing such a reagent moves, the light emitted from the blue light source 67 irradiates the nitrocellulose membrane 51 in the vicinity of the second reagent holding portion 53, so that the sample solution containing the reagent becomes a blue light source. When passing through the portion irradiated with light from 67, the amount of light reaching the detector 69 that detects the light from the blue light source 67 changes due to light absorption by the phenanthroline iron complex dissolved in the sample solution.

  When the detection signal from the detector 69 falls below the threshold value I, a substrate liquid release determination process, which will be described later, is performed according to a command from the calculation unit 101. It is compared with the minimum value stored in the memory 104, and a smaller value is overwritten and stored in the memory 104 as the minimum value. When there is no minimum value stored in the memory 104, a numerical value lower than the threshold value I is stored as the minimum value.

In the substrate liquid release determination process, the detection signal is also compared with a threshold value II stored in advance in the ROM 105, and when the numerical value is larger than the minimum value and larger than the threshold value II, the test piece 2 is detected by a signal from the calculation unit 101. Several volts between the carbon electrode portions 45b and 46b and the carbon electrode portions 45c and 46c of the test piece 2 through the connection between the connection portions 43a and 44a of the PTFE substrate 41 and the connector portion 72 of the measuring device 3 respectively. Is applied. Thereby, the substrate solution for the enzyme reaction, that is, 3,3′-diaminobenzidine contained in the first electroresponsive gel and H ₂ O ₂ contained in the second electroresponsive gel are released.

  Thus, since the reflected light of the light emitted from the blue light source 67 is received by the detector 69, the mixed reagent of the first antibody, enzyme-supporting insoluble particles and dye passes through the second reagent-supporting portion 53 in a non-contact manner. By detecting this, the optical measuring unit 66 is hardly contaminated during measurement, and maintenance of the measuring device 3 is facilitated.

By the substrate solution release determination process, the sample solution containing the reagent passes through the vicinity of the second reagent holding portion 53 and is then contained in the substrate solution for the enzyme reaction, that is, the first electroresponsive gel. '- it is possible to release H ₂ O ₂ contained in diaminobenzidine and second electrically responsive gel.

  Although the timing of release depends on the setting of the threshold value II described above, if the threshold value II is set to a value close to the minimum value that can be taken when the test piece 2 is normal by a prior trial, the sample liquid containing the reagent is The substrate liquids can be released immediately after passing through the vicinity of the second reagent carrying part 53.

  The carbon electrodes 45b and 46b and 45c and 46c of the test piece 2 are connected to the connector part 72 of the table part 64 through the connection between the connection parts 43a and 44a of the test piece 2 on the PTFE substrate 41 and the signal from the calculation unit 101. A voltage of several volts is applied between the first electroresponsive gel 48 and the second electroresponsive gel 49. Due to the contraction of the first electro-responsive gel 48 and the second electro-responsive gel 49, each of the released substrate liquids moves through the nitrocellulose membrane 51 by capillary action and is captured by the second reagent carrier 53. Reacts with the enzyme on the polystyrene latex particles to deposit reddish brown reactants.

  As the reddish brown reactant is deposited, the amount of light detected by the detector 69 changes. When the signal change due to the change in the light amount becomes 5% or less per 10 s by the calculation in the calculation unit 101, the signal acquisition from the detector 69 by the calculation unit 101 is temporarily stopped.

  And the drive mechanism part 65 is operated with the pulse signal which generate | occur | produced by the instruction | command from the calculating part 101, and the table part 64 is moved to the position set beforehand.

  After the table 64 is moved to the set position, the drive mechanism 65 is actuated again by the pulse signal generated by the command from the calculation unit 101, and the blue light source 67 is moved on the second reagent holding unit 53 of the test piece 2. The table part 64 moves so that the emitted light from the light traverses. Simultaneously with the command to the drive mechanism unit 65, the calculation unit 101 resumes signal acquisition from the detector 69.

  The fetched signal data from the detector 69 is associated with the movement distance of the table unit 64 calculated by the calculation unit 101 from the pulse count result of the pulse counter 106 with respect to the pulse signal to the drive mechanism unit 65, and stored in the memory 104. Saved in a new storage area.

  The data about the movement distance of the table unit 64 and the detected light amount change of the detector 69 obtained as described above is integrated with respect to the distance by the calculation unit 101, and the value obtained by the integration is measured and stored in the memory 104. To do. From this measured value and a calibration curve previously stored in the ROM 105, the antigen concentration can be obtained by calculation in the calculation unit 101. The obtained result is stored in the memory 104. The obtained result is displayed on the display unit 5 by the display mechanism unit 102 according to a command from the calculation unit 101 to notify the user.

  In this way, by displaying on the display unit 5, it is possible to notify the user of the operation procedure and to display the measurement result, so that the operability of the immunochromatographic measurement system can be increased.

  As described above, according to the present embodiment, it is possible to perform immunochromatographic measurement sensitized by an enzyme just by applying a sample solution to an immunochromatographic test piece, and the user does not have specialized knowledge about analytical techniques. Even if you are unfamiliar, you can easily make correct measurements.

  The immunochromatographic test strip of the present invention and the immunochromatographic measurement method using the same are useful for measuring the amount of a specific substance contained in a collected body fluid.

The perspective view which shows the external appearance of the immunochromatography measuring system in embodiment of this invention The perspective view which shows the test piece which comprises the same immunochromatography measuring system (A) is a side view showing a test piece constituting the immunochromatography measurement system, and (b) is a plan view showing a test piece constituting the immunochromatography measurement system. Plan view showing a part of the preparation process of the test piece constituting the immunochromatographic measurement system Plan view showing the continuation of the test piece production process Partially cutaway front view of the measuring device constituting the immunochromatographic measurement system AA sectional view in FIG. 6 of the measuring apparatus constituting the immunochromatographic measuring system Sectional view of the immunochromatography measurement system Sectional view of the immunochromatography measurement system Operation timing diagram of the immunochromatography measurement system Block diagram of the processing control unit of the immunochromatography measurement system

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Immunochromatography measurement system 2 Test piece 3 Measuring apparatus 4 Main power supply 5 Display part 6 Measurement start button 7 Test piece insertion port 21 Upper cover 21a Sample liquid injection port 21b Measurement window 21c Air hole 22 Lower cover 23 Inspection base body 41 PTFE board 42 Wiring portion 43a, 44a Connection portion 43b, 43c, 44b, 44c Electrode portion 43d, 44d Lead portion 45b, 45c, 46b, 46c Carbon electrode portion 47 Insulating film 48 First electric responsive gel 49 Second electric responsiveness Gel 51 Nitrocellulose membrane 52 First reagent carrying part 53 Second reagent carrying part 54 Absorbent pad 55 Sample liquid filter paper 60 Processing control part 61 Apparatus housing 62 Measurement chamber housing part 63 Housing 63a Housing upper part 63b Side surface Part 63c Reinforcing part 63d Optical measurement mounting part 63e Groove 64 Table part 65 Drive mechanism 65a Stepping motor 65b Male thread portion 66 Optical measurement portion 67 Blue light source 68 Optical slit 69 Detector 71 Table body 71a Holding surface 71b Guide surface 71c Side surface portion 71d Projection portion 71e Recessed portion 72 Connector portion 72a Contact piece 72b Housing 73 Followed screw portion 74 Bearing Housing 75 Female screw nut part 101 Calculation part 102 Display mechanism part 103 Timekeeping part 104 Memory 105 ROM
106 Pulse counter

Claims (2)

An immunochromatographic test piece for measuring an antigen in a sample solution,
Insulating substrate,
A pair of electrodes disposed on the substrate;
An electroresponsive gel disposed on the pair of electrodes and holding a solution containing a substrate for the enzyme;
A porous member disposed on the substrate at a position in contact with the electrically responsive gel;
A reagent part disposed on the porous member so that a reagent containing particles and a dye supporting the first antibody and the enzyme carrying the antigen moves with the sample solution;
A second antibody capable of binding to a site different from the first antibody, which is an antibody against the antigen, and is disposed at a position in contact with the porous member; A water absorption part capable of absorbing the sample liquid,
An immunochromatographic test in which the reagent part, the immobilization part and the water absorption part are arranged so that the sample solution supplied onto the porous member flows in the order of the reagent part, the immobilization part and the water absorption part. Piece. Using the immunochromatographic test piece according to claim 1,
(A) supplying the sample solution onto the porous member;
(B) a step of detecting that the sample solution containing the reagent has passed through the immobilization unit;
(C) a step of automatically applying a voltage between the pair of electrodes based on the detection in the step B;
(D) a step of detecting the amount of the reaction by the enzyme in the immobilization part, and (E) a step of determining the concentration of the antigen contained in the sample solution based on the detection amount in the step D .

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